Circulating virome and inflammatory proteome in patients with ST-elevation myocardial infarction and primary ventricular fibrillation

Primary ventricular fibrillation (PVF) is a life-threatening complication of ST-segment elevation myocardial infarction (STEMI). It is unclear what roles viral infection and/or systemic inflammation may play as underlying triggers of PVF, as a second hit in the context of acute ischaemia. Here we aimed to evaluate whether the circulating virome and inflammatory proteome were associated with PVF development in patients with STEMI. Blood samples were obtained from non-PVF and PVF STEMI patients at the time of primary PCI, and from non-STEMI healthy controls. The virome profile was analysed using VirCapSeq-VERT (Virome Capture Sequencing Platform for Vertebrate Viruses), a sequencing platform targeting viral taxa of 342,438 representative sequences, spanning all virus sequence records. The inflammatory proteome was explored with the Olink inflammation panel, using the Proximity Extension Assay technology. After analysing all viral taxa known to infect vertebrates, including humans, we found that non-PVF and PVF patients only significantly differed in the frequencies of viruses in the Gamma-herpesvirinae and Anelloviridae families. In particular, most showed a significantly higher relative frequency in non-PVF STEMI controls. Analysis of systemic inflammation revealed no significant differences between the inflammatory profiles of non-PVF and PVF STEMI patients. Inflammatory proteins associated with cell adhesion, chemotaxis, cellular response to cytokine stimulus, and cell activation proteins involved in immune response (IL6, IL8 CXCL-11, CCL-11, MCP3, MCP4, and ENRAGE) were significantly higher in STEMI patients than non-STEMI controls. CDCP1 and IL18-R1 were significantly higher in PVF patients compared to healthy subjects, but not compared to non-PVF patients. The circulating virome and systemic inflammation were not associated with increased risk of PVF development in acute STEMI. Accordingly, novel strategies are needed to elucidate putative triggers of PVF in the setting of acute ischaemia, in order to reduce STEMI-driven sudden death burden.

Statistical analysis. Summary data were represented by mean and standard error of the mean (SEM), or by median and interquartile range (IQR) depending on the data normality. The D' Agostino and Pearson test was used to evaluate the normality of data. Two-groups comparisons were performed using the unpaired t-test or Mann Whitney test, and three-groups comparisons were performed using Kruskal-Wallis test or ANOVA, depending on the data normality. Fisher's exact test was used when required. Statistical significance was assumed www.nature.com/scientificreports/ when P was < 0.05. Statistical analyses were performed using Prism 9 for macOS version 9.0.2 (134) and 9.3.1 (350).

Results
Circulating virome. The virome capture sequencing platform VirCapSeq-VERT was used to target viral taxa in human blood samples from non-PVF patients (n = 9) and PVF patients (n = 11). The capture results were sequenced using Illumina HiSeq/NovaSeq. Human and bacterial reads were removed from the sequencing files, and the remaining reads ranged from 148-543 k pairs of reads per sample (Supplementary Table 1). Along all reads and samples, we found good quality per base position. However, we detected a high amount of PCR duplicates, due to the amplification and enrichment protocol (Supplementary Table 2). We also identified and removed common sequencing adapters. Details in the statistics regarding the trimming process for each sample are shown in Supplementary Table 3. The host depleted trimmed reads were assembled using SPAdes software to generate longer sequences, and for an additional and improved homology search (Supplementary Table 4). All viral reference genomes available in GenBank NCBI database were used to create a BLAST database, which was used for the homology search, with the generated assembled host depleted trimmed reads as input. We observed a total of 51 different genome entries ( Table 2). The identified genome entries were then used to create an alignment index, to map the reads corresponding to their exact position in the reference genomes. Nearly half of the reads did not map to retrieved viral sequences, likely because the host depleted reads may have contained archaea, yeast, or unclassified taxon reads. These results correspond with the low number of reads identified as viral (Supplementary Table 1). Supplementary Tables 5 and 6 summarize the length of each reference sequence, and the percent of base pairs covered (%), in non-PVF and PVF patients. Six genome entries found in the homology search did not generate reads mapping, since the better sensitivity of the mapping enabled more confident placing of a read compared to with BLAST.
Alpha diversity. We further assessed alpha diversity to determine the diversity and to enable comparisons of the type and quantity of virus species between non-PVF and PVF patients. Alpha diversity is a statistic used in this kind of sample, in which reads reflect the abundance of each of the identified operational taxonomical units (OTUs). Richness and diversity are alpha diversity metrics.
IgG-class antibodies to Epstein-Barr nuclear antigen were detected in 94.8% of the analyzed population. Similar to the non-significant differences found in Human gammaherpesvirus 4 (NC_007605.1) frequencies between non-PVF and PVF patients by sequencing, IgG-class antibody detection levels did not significantly differ between non-PVF and PVF patients (31.42 ± 1.121 vs. 33.40 ± 1.125 Units; P = 0.196) (Fig. 5B).

Discussion
Primary ventricular fibrillation (PVF) is among the leading causes of prehospital sudden cardiac death. It is presently unknown what factors increase the probability of PVF development during acute ischemia, complicating the identification of PVF predictors. We thus aimed to evaluate possible PVF predictors or triggers, including the complete DNA virome and the inflammatory proteome in PPCI-treated STEMI patients. A growing number of viruses have been determined to be associated with inflammatory cardiomyopathy. Previous data suggest that viral exposure could increase PVF susceptibility, although this has not been conclusively proven. In this context, Andréoletti et al. identified coxsackievirus B infection in post-mortem endomyocardial tissue of patients who died suddenly due to AMI 10 . Additionally, the AGNES (Arrhythmia Genetics in the NEth-erlandS) study showed that PVF during first STEMI was most significantly associated with SNP rs2824292 at www.nature.com/scientificreports/ chromosome 21q21, where the CXADR gene is found. CXADR encodes the coxsackie and adenovirus receptor protein, which has been implicated in myocarditis 28 , dilated cardiomyopathy 28 , and ventricular conduction and arrhythmia vulnerability 29 . However, this association was not replicated in at least two additional studies 30,31 . Extreme influenza epidemics are also reportedly associated with out-of-hospital cardiac arrest 8 . However, no other relationships have been found between PVF occurrence and enterovirus or influenza exposure 9 . The present pilot study is the first to include a circulating virome analysis of all DNA viruses that infect vertebrates. Our findings indicate that non-PVF and PVF patients significantly differed only in the levels of Macacine gammaherpesvirus 4 (Rhesus lymphocryptovirus), Human herpesvirus 4 type 2 (Epstein-Barr virus type 2), and Torque teno viruses 8 and 18 (transfusion transmitted viruses).
Gamma-herpesvirinae family viruses are lymphotropic viruses that infect lymphoid cells. Epstein-Barr virus (EBV) is a highly ubiquitous herpesvirus which asymptomatically infect over 90% of the population 32 . Once infected, EBV persists in B-cells for life and could be reactivated in immunosuppression cases 33 . In terms of the heart, EBV reportedly induces severe infection of T-cells in the myocardium of patients with ongoing myopericarditis 34,35 , as well as in abdominal or coronary aneurysms 36,37 . EBV infection may also influence the development of atherosclerosis 38 . Here we identified EBV (Human gammaherpesvirus 4) and EBV type 2 (Human herpesvirus 4 type 2). The relative frequency of EBV did not significantly differ between non-PVF and PVF patients. Along this line, we did not find significant differences in the viral load or in the IgG-class antibodies to EBV between non-PVF and PVF patients measured by RT-PCR and ELISA, respectively. On the other hand, the EBV type 2 frequency was significantly higher in non-PVF patients than in PVF patients, and is thus not a risk factor for second-hit ischaemia-driven cardiac arrest. Any of the patients analysed took immunosuppressive treatment or had any malignancy.
Furthermore, Torque teno viruses (TTVs) are small DNA viruses that have been detected in many mammalian hosts, and whose prevalence in humans is > 90% 39 . It is not clear that TTVs act as primary pathogens, and it appears that TTVs usually establish chronic infections without causing pathology. It has been suggested that TTVs could be used as markers of viral environmental contamination, since TTVs are potential contaminants www.nature.com/scientificreports/ in water sources 40 and hospitals 41 , including in the blood supply 42 . This may explain why we detected 27 species of TTVs in the presented study. Remarkably, among 20 human samples, only 1 tested negative for all detected TTV species. Although they are not among the 10 most frequent relative entries, TTV-8 and TTV-18 were the most frequently detected TTVs, and their frequencies significantly differed between non-PVF and PVF patients. However, the relative frequencies of TTV-8 and TTV-18 were significantly higher in non-PVF patients than in PVF patients, and thus do not provide information to predict sudden cardiac arrest. Takeuchi et al. detected one TTV sequence read in a patient with acute myocarditis, but could not establish it as a potential pathogen of myocarditis 43 . Both our results and Takeuchi's findings support the widespread idea that TTVs are unlikely to act as primary pathogens.
The second objective of this study was to examine the systemic inflammatory response, which is known to play important roles in the pathophysiology of acute coronary syndrome and atherosclerosis. Notably, in recent years, its involvement in SCD has also been studied, although attempts to find predictive biomarkers have yielded inconclusive results 44 . The Physicians' Health Study showed that C-reactive protein (CRP) levels are an independent risk factor for SCD (OR, 2.78; 95% CI, 1.35-5.72) 45 . In contrast, the Nurses' Health Study did not confirm any significant correlation between SCD and highly sensitive CRP 46 . Among healthy European middle-aged men who participated in the PRIME Study, higher IL-6 was a strong predictor of sudden death, with an OR of 3.06 (95% CI, 1.20-7.81) 11 , but CRP was not shown to predict SCD, as in the Nurses' Health Study. Furthermore, our group identified growth differentiation factor 15 (GDF-15) as a predictor of mortality and CV morbidity 47 , and Andersson et al. detected GDF-15 as a risk factor for sudden cardiac death in the acute phase of MI, with an OR of 1.47 (95% CI, 1.11-1.95) 12 .
Our analyses revealed no significant differences between non-PVF and PVF patients for any of the analysed inflammatory-related proteins. We did identify differential protein expression between healthy subjects and STEMI patients (including both non-PVF and PVF patients) ( Supplementary Fig. 5). Compared to healthy subjects, STEMI patients showed significantly higher levels of inflammatory proteins related to cell adhesion, chemotaxis, and cellular response to cytokine stimulus, and cell activation proteins involved in immune response, such as IL-6, IL-8 CXCL11, CCL11, MCP3, MCP4, and ENRAGE. The roles of IL-6 and IL-8 in AMI have been previously described 48,49 . CCL11 has potent eosinophil chemoattractant activity, and is expressed by cardiac macrophages 50 . Here we found that CCL11 levels were increased in STEMI patients compared to healthy subjects, thus confirming the previously observed association between CCL11 and myocardial infarction 51,52 . MCP-3 plays an important role in cell recruitment to inflammatory sites, specifically, it has been described that MCP-3 recruits mesenchymal stem cells and improved cardiac remodeling 53 . Mao et al., found that MCP-3 levels were decreased in patients with cardiac remodeling after AMI compared to MI and control groups; in addition, MCP-3 values were not differential between MI and healthy subjects 54 . These results do not agree with what was found in our pilot study, so delving into the role that MCP-3 plays in STEMI patients would be interesting.
Although no differences were found between non-PVF and PVF patients, some proteins significantly differed between the healthy control group and one of the STEMI groups. For example, CDCP1 and IL18-R1 were significantly higher in PVF patients than in healthy subjects. Shia et al. conducted genome-wide association analyses, and identified variations in the DNA sequence that affect the expression of 3p21.31 (CDCP1), which were associated with myocardial infarction 55 . Those authors did not specify whether the patients had PVF. In the other hand, Ponasenko et al. also found that a polymorphic variant of IL18R1 was associated with an increased risk of MI in CAD patients with coronary artery disease 56 . Based on our results, it would be interesting to further examine into the studies related to CDCP1 or IL18-R1 and PVF. In contrast, MCP-1, CCL4, TNFRSF-9, and NT-3 showed different expression profiles in healthy subjects compared to non-FVP patients, but not compared with FVP patients. The association of some of them with cardiovascular disorders has already been previously described by other authors. MCP-1, which recruit circulating monocytes, plays a major role in the immunologic profile of ischaemia/reperfusion injury in the heart 57 ; CCL4 is directly involved in the atheroma plaque stabilization 58 ; and elevated NT-3 plasma levels are associated with an increased risk atrial fibrillation recurrence 59 . However, there remains a need to elucidate potential key roles of these proteins in inflammatory process development in AMI; and they do not seem to be involved in PVF.
This study has several limitations. It was a pilot study with a limited sample size. Despite comprehensive examination of both the virome and the proteome, we did not identify any clear trend. The VirCapSeq-VERT panel can capture both DNA and RNA viruses; however, due to the storage conditions and available blood material, we cannot fully exclude the presence of undetected RNA viruses. In addition, we have not been able to make the correlation between the OTUs and the inflammatory protein levels because, although the population is the same, some samples were used to the virome screening study and others to the inflammation analyses. Lastly, to confirm the presence of a viral genome within the myocardium during the acute phase of STEMI, we would need to perform endomyocardial biopsies, which is ethically unacceptable.
In conclusion, our observations revealed no clear trend in associations between the circulating virome or inflammatory proteome and PVF in STEMI. Hence, there remains a critical need for new strategies to better elucidate the possible triggers of PVF, and to identify individuals at high risk of SCD.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.